Neomycin and Clotrimazole Loaded Chitosan-Based Electrospun Wound Dressing: Development, Characterization, and Antimicrobial Properties

Abstract

This study reports a novel approach to chronic wound therapy by fabricating a dual antimicrobial electrospun wound dressing that simultaneously delivers neomycin (antibacterial) and clotrimazole (antifungal) in a poly­(vinyl alcohol) (PVA) and biocompatible chitosan (CS) PVA/CS matrix. Compared to single-drug products in use today, this new dressing mitigates polymicrobial infections characteristic of chronic wounds through controlled dual-drug release kinetics, with initial rapid neomycin (NM) release to instantaneously control bacteria and slow diffusion of clotrimazole (CZ) to provide extended antifungal coverage. Because they take longer to heal, chronic wounds with bacterial and fungal infections pose serious healthcare difficulties. The optimal polymer combination of 3% (w/v) PVA and 3% (w/v) chitosan in a ratio of 4:1 (PVA/CS) was determined, with drug concentrations being 0.5% (w/v) neomycin and 1% (w/v) clotrimazole, and nanofibers with average diameters of 60 ± 18 nm (control) and 87 ± 26 nm (drug-loaded films) were achieved. The dressings reported good mechanical properties (7.49 MPa tensile strength) and enhanced fluid absorption capacities (299% water absorption), suitable for application in wound healing. Kinetic patterns in drug release investigations revealed therapeutic activity optimized uniquely. Neomycin displayed rapid initial release (40% within the first hour) following non-Fickian Weibull kinetics (R ² = 0.88), while clotrimazole showed sustained Higuchi-type diffusion (R ² = 0.97). The wound dressing exhibited greater antimicrobial activity against Gram-positive and Gram-negative bacteria (zones of inhibition: 22.5 ± 0.7 mm and 18.5 ± 0.7 mm, respectively) and excellent antifungal activity against Aspergillus niger and Lasiodiplodia theobromae (zones of inhibition: 35.8 ± 7.4 mm and 27.0 ± 1.4 mm, respectively). The FTIR analysis confirmed successful drug incorporation within the polymer matrix. This innovative multifunctional electrospun wound dressing is effectively fabricated with tailored dual antimicrobial release profiles, achieving an integrated approach to chronic wound treatment. Subsequent studies should involve in vivo investigations and clinical trials for evidence of safety and efficacy in the future.

1. Introduction

The skin is the biggest organ in the human body, which is a barrier between the body’s internal organs and the outside world. The average adult’s skin weight is 15% of their total body weight. It is considered the crucial element that shields inside tissues from severe weather, UV rays, microbial infections, and mechanical trauma. , A wound can be defined as an alteration to the mucosal surface, skin, or an organ’s tissue within the body. Any area of the body can be subject to injuries due to an accident, an illness, or an intentional cause due to disturbance caused by microbes, heat, matter, or chemicals. , In order to treat such wounds, drugs are delivered by some parenteral pathways, such as intramuscular, subcutaneous, intravenous, or enteral pathways, in the form of tablets, capsules, and granules. These drug delivery methodologies have some drawbacks, such as first-pass metabolism, fatigue, or discomfort. As a method to overcome those situations, drugs are applied to the skin directly. In the context of acute wounds, the healing process is a complex biological process involving four main stages: hemostasis, inflammation, proliferation, and maturation. Delayed healing of an acute wound might lead to the possible development of chronic wounds. As with requirement and demand, different types of biomaterials have become an essential class of materials worldwide. Among the novel trends in biomaterials, the development of advanced wound dressings is a main field of research that has interested many researchers. Advanced dressings such as films, foams, hydrogels, hydrocolloids, and drug-loaded products have been developed to promote the healing of chronic wounds over traditional dressings. , These novel substances have benefits such as enhancing hemostasis, controlling microbial growth, and accelerating healing. Effective control of microbial growth also prevents acute wounds from becoming chronic. ,, Advanced wound dressings combine essential functions to fulfill customized patient needs, which improves wound care.

Chitosan (CS) and poly­(vinyl alcohol) (PVA) are a perfect polymer combination for advanced wound dressing applications because of their shown biocompatibility and complementary qualities. The properties such as, excellent film-forming qualities, high tensile strength, and enhanced processability during electrospinning are all provided by PVA, a synthetic water-soluble polymer. Its hydrophilic properties allow for efficient fluid absorption, which is essential for managing exudate during wound healing. Direct skin contact is also acceptable due to PVA’s nontoxic nature and FDA approval for biomedical purposes. Chitosan, derived from the deacetylation of chitin, shows unique biological advantages on the formulation. As chitosan is the second most abundant biopolymer in nature after cellulose, and also, the only natural positively charged polysaccharide, it has a natural antimicrobial property against fungi and bacteria through electrostatic interaction with negatively charged microbial cell walls. , Its biocompatibility, biodegradability, and hemostatic activity promote natural wound healing processes. Chitosan also possesses excellent mucoadhesive properties, which allow prolonged contact time at wound surfaces to ensure drug delivery. , With the above attributes discussed, it is possible to state that the combination of PVA/CS makes an optimum environment for drug delivery. This combination offers improved properties for a wound dressing, where CS provides biological activities and controlled drug release, and PVA contributes to the fabrication and structural integrity of the wound dressing. , With that, this polymer blend was chosen for this study to address a few different needs for wound care management, such as better fluid absorption, biocompatibility, sustaining of antimicrobial properties, and sufficient mechanical strength.

Electrospinning is a technique of fabricating man-made fibers using electrostatic forces. This technique, known earlier as “electrostatic spinning”, was discovered long ago. Cooley and Morton initially patented electrospinning in 1902 and gave details of the apparatus for electrically dispersing fluids. , An electrospinning setup consists of three main components: a pump, a collector, and a high-voltage generator. The applications of electrospinning have spread to a large area in diverse industries. Electrospinning can be widely seen in the manufacturing of cosmetics, the textile industry, electrical applications, renewable energy, biomedical applications, and filtration processes. , In the context of wound dressings, for the fabrication process, electrospinning has been widely applied, especially in polymer-based wound dressings. The incorporation of various drugs, nanoparticles, and plant extracts has been recorded. − Some mandatory requirements of wound dressings, such as fluid absorption and gas transmission, are inherently incorporated by using an electrospinning procedure.

Combining multiple requirements within a single product will be an ideal solution for treating chronic wounds. Modern dressings can be customized to meet the unique needs of patients and include drug micropropagation for effective healing. These dressings improve physical characteristics for patient comfort and accelerate healing by loading artificial or natural drugs, such as metal nanoparticles, antibiotics, antioxidants, and nonsteroidal anti-inflammatory agents , Neomycin sulfate and clotrimazole are two antibacterial and antifungal active pharmaceutical ingredients (APIs) that are commonly used in dermatological ointments. , It is assumed that the incorporation of drugs into the electrospun films will bring several advantages over ointments, such as improved wound healing, ease of use directly, and less waste of drugs, as the drugs will be directly absorbed into the wound from the dressing. This study aims to develop a multifunctional wound dressing by using two polymers (PVA and CS) and incorporating two antibacterial and antifungal drugs using an electrospinning synthesis method.

Therefore, the fabrication of the wound dressing of this study is done with the PVA–CS polymer blend, and a combination of drugs is loaded into this polymeric wound dressing. For that, neomycin and clotrimazole are incorporated into this electrospun matrix to address a few critical gaps in the current stage of wound care technology. , Neomycin, which is known to be a broad-spectrum antibiotic, indicates superior efficacy against common bacterial groups in wound infections. , These common wound pathogens include Escherichia coli and Staphylococcus aureus, which are frequently found in wounds, and previous studies also prove that topical neomycin-related applications improve wound healing by decelerating bacterial growth, along with reduced risks of toxicity. , Strong electrostatic interactions with negatively charged bacterial surfaces are made possible by the neomycin’s polycationic nature at physiological pH, which promotes membrane permeabilization and intracellular accumulation. Clinical evidence supports neomycin’s efficacy in wound applications, with studies demonstrating that neomycin, both alone and in combination with other antimicrobials, restricted the growth of resistant organisms, indicating that combination therapy may be useful in the prevention and/or treatment of complicated wound infections. Clotrimazole is an antifungal agent that belongs to the type of imidazole and operates by blocking the cytochrome P450-dependent enzyme 14α-lanosterol demethylase, which is essential for the formation of fungal ergosterol. This process compromises the integrity of the fungal cell membrane, increasing membrane permeability and ultimately causing cell death. According to clinical applications, the most often used treatment for fungal infections is a 1% of clotrimazole, which demonstrates a better action against Aspergillus and Candida species. This formulation’s choice of 1% w/w concentration ensures compatibility with the polymer matrix and sustained release properties while also being in line with recognized therapeutic doses. The neomycin-clotrimazole combination addresses the underlying dilemma of polymicrobial wound infection. Unlike sequential single-agent treatments, combined therapy permits simultaneous protection against both bacterial and fungal pathogens, along with the exploitation of synergistic mechanisms of action.

Despite the documented advantages of electrospun wound dressings, a critical gap remains in developing comprehensive antimicrobial systems that simultaneously address both bacterial and fungal infections, the predominant causes of polymicrobial wound complications. Therefore, this study aims to develop and characterize a dual-drug-loaded electrospun wound dressing by incorporating neomycin (antibacterial) and clotrimazole (antifungal) into a PVA–chitosan nanofiber matrix fabricated via electrospinning technology. The novelty of this work lies in the simultaneous incorporation of both antibacterial and antifungal agents within a single electrospun PVA–chitosan nanofiber platform, representing a unique approach to addressing the complex polymicrobial nature of wound infections through one biocompatible delivery system. This multifunctional wound dressing leverages the synergistic benefits of chitosan’s inherent antimicrobial properties, the sustained release characteristics of electrospun nanofibers, and the biomimetic nanofibrous architecture that mimics the native extracellular matrix to promote cell adhesion and tissue repair while maintaining optimal moisture balance and gas exchange. The findings of this research contribute to advancing next-generation wound care materials by providing a clinically relevant therapeutic platform that offers broad-spectrum antimicrobial protection, improved patient compliance through reduced administration frequency, and enhanced healing outcomes for complex wound management.

2. Results and Discussion

2.1. Optimization of Electrospinning Parameters to Synthesize Polyvinyl Alcohol/Chitosan (PVA/CS) Nanofibers

Optimizing the voltage parameter was tried first by keeping other parameters constant. A voltage range from 15.6 kV to 26.6 kV was used, and the best fiber formation was gained at 21 kV. Initially, polymer suspension parameters were conducted with their electrospinning parameters (see Supporting Information, Tables S1 and S2). Next, the concentration was changed and observed spinning to adjust the electrospinning parameters. Initial polymer suspension was optimized by electrospinning poly­(vinyl alcohol) (PVA) 6% (w/v) neat films with only changing flow rates (FR) from 0.2 mL/h to 1.0 mL/h to optimize the flow rate. Higher flow rates showed higher droplets spotting on the film, where it produces numerous beads. With that, the flow rate was optimized to 0.2 mL/h. The tip-to-collector distance (TCD) was adjusted on the final occasion, where a range of distance from 7 to 16 cm was used. With the optimization process, a TCD of 9 cm was fixed as it provided a well-formed fiber film, which was easily removed from the aluminum foil. The neat PVA 6% (w/v) film’s fibers were formed in a better way with a fiber diameter of 115 ± 36 nm. Then, with the optimized electrospinning parameters, PVA/chitosan (CS) films were electrospun. In that instance, the optimized voltage of 21 kV was applied, and by that, bead-free fibers were obtained. A few other films were fabricated with the optimized PVA/CS polymer concentrations and electrospinning parameters.

2.2. Characterization of Nanofibers

2.2.1. Fourier Transform INFRA-RED (FT-IR) Spectroscopy

The comparison of FT-IR spectra of PVA, CS, NM, CZ, and PVA–CS–NM–CZ–film is shown in (Figure ). Functional groups of O–H stretching at 3276 cm^–1, C–H stretching at 2942 cm^–1, CH₂ symmetric stretching at 2908 cm^–1, CH₂ bending at 1442 cm^–1, and C–O stretching at 1145 cm^–1 were identified from PVA. Functional groups of N–H stretching at 3370 cm^–1, C–H stretching at 2871 cm^–1, N–H bending at 1595 cm^–1, and C–O stretching at 1062 cm^–1 were identified from CS. Functional groups N–H Stretching at 3361 cm^–1 O–H stretching at 3214 cm^–1, C–H stretching at 2899 cm^–1, CC Stretching at 1622 cm^–1, CC stretching (aromatic) at 1521 cm^–1 and C–O at 1032 cm^–1 were identified from NM, and functional groups of C–H Stretching (Aromatic) at 3061 cm^–1, CC Stretching at 1583 cm^–1, C–N Stretching at 1209 cm^–1, C–N Stretching at 1041 cm^–1, C–Cl Stretching at 827 cm^–1 were identified from CZ. All the identified peaks and their functional groups are listed in (Table ). The successful incorporation of NM, CZ, PVA, and CS in the wound dressing is confirmed by the FT-IR spectrum of PVA–CS–NM–CZ film. In the FTIR spectrum of the drug-loaded film shows the main functional groups as O–H stretching (Water), C–H stretching, CC stretching, CC stretching (Aromatic), CH₂ bending, C–N stretching, and C–Cl stretching. It confirms the integration of PVA, CS, NM, and CZ by these functional groups. O–H stretching (water), CH₂ bending by PVA, C–H stretching by PVA or CS, CC stretching (aromatic) by NM or CZ, CC stretching by NM, C–Cl stretching, and C–N stretching by CZ.

1.

FT-IR spectra of (a) drug-loaded film (PVA–CS–NM–CZ–film), (b) neomycin, (c) clotrimazole, (d) chitosan, and (e) PVA.

1. FT-IR Wavenumbers and Functional Groups.

substance	observed wavenumber (cm–1)	characteristic range (cm–1)	functional group	references
PVA	3276	3550–3200	O–H stretching	,
	2942	2990–2850	C–H stretching
	2908	2800–3000	CH2 symmetric stretching
	1442	1470–1430	CH2 bending
	1145	1300–1000	C–O stretching
CS	3370	3550–3250	N–H stretching
	2871	2990–2850	C–H stretching
	1595	1600–1500	N–H bending
	1062	1080–1030	C–O stretching
NM	3361	3550–3250	N–H stretching	,
	3214	3550–3200	O–H stretching
	2899	2900–2800	C–H stretching
	1622	1680–1620	CC stretching
	1521	1625–1440	CC stretching (aromatic)
	1032	1300–1000	C–O
CZ	3061	3100–3000	C–H stretching (aromatic)
	1583	1625–1440	CC stretching (aromatic)
	1209	1250–1000	C–N stretching
	1041	1250–1000	C–N stretching
	827	600–840	C–Cl stretching
PVA–CS–NM–CZ Film	3291	3550–3200	O–H stretching (water)	–
	2908	2990–2850	C–H stretching
	1638	1680–1620	CC stretching
	1546	1625–1440	CC stretching (aromatic)
	1433	1430–1470	CH2 bending
	1064	1250–1000	C–N stretching
	823	1250–1000	C–Cl stretching

2.2.2. Scanning Electron Microscopy

Optimization of both polymer suspension and electrospinning parameters was done by analyzing the SEM images. The initial SEM images confirmed that the fibers were full of beads, and gradually, they developed into fibers. The clear fibers were obtained from PVA 6% neat film, and the 6% solid loading was kept constant and incorporated CS as PVA 3% (w/v) + CS 3% (w/v). However, it is observed that the incorporation of drugs makes beads in the fibers (supplementary section Figures S1 and S2) The average fiber diameter of neat PVA fibers has an average diameter of 115 ± 36 nm and PVA 3% (w/v) + CS 3% (w/v) results in an average diameter of 87 ± 26 nm. At the polymer concentration of PVA 3% (w/v) + CS 1.5% (w/v), the morphology has changed to a structure of fibers with beads. Those fibers have an average diameter of 46 ± 11 nm. With the addition of the drug, the average fiber diameter was increased to 60 ± 18 nm, with some bead formation. The SEM images of PVA 6% (w/v), PVA 3% (w/v) + CS 3% (w/v), PVA 3% (w/v) + CS 1.5% (w/v), PVA 3% (w/v) + CS 3% (w/v) + NM 0.5% (w/v) + CZ 1% (w/v) and their average fiber diameter histograms are shown in Figure . This study shows a PVA concentration of 6% (w/v) (PVA neat) shows the best fiber formation with an average FD of 115 ± 36 nm, whereas a similar study has reported an average fiber diameter (FD) of 240–300 nm for electrospun fibers of PVA and chitosan which is higher than the value of this study. A combination of CS 3% (w/v) and PVA 3% (w/v) has also yielded a fiber with moderate diameter distribution with an average FD of 87 ± 26 nm. The coefficient of variation (CV) for the drug-loaded PVA–CS–NM–CZ formulation was calculated to be 29.88% (CV = standard deviation/mean × 100 = 26/87 × 100), indicating a moderate fiber diameter distribution. While CV values between 20% and 40% are commonly reported for drug-loaded electrospun fibers, this variation reflects the presence of multiple components that affect jet behavior during electrospinning. The incorporation of both hydrophilic (neomycin) and hydrophobic (clotrimazole) drugs creates localized variations in solution properties, leading to heterogeneous fiber formation. Similar observations have been reported in other multicomponent electrospun systems, where drug loading influences fiber diameter distribution. Despite this variation, the nanofiber structure remains functional for wound healing applications, maintaining adequate porosity and mechanical properties. Johnson et al. demonstrated that the incorporation of therapeutic drugs into electrospun poly-l-lactic acid fibers resulted in decreased fiber diameter and altered fiber alignment, indicating that drug incorporation significantly affects fiber physical properties.

2.

SEM Images of (a) PVA 6%, (b) PVA 3% + CS 3%, (c) PVA 3% + CS 1.5%, (d) PVA 3% + CS 3% + NM 0.5% + CZ 1% and average fiber diameter histograms of (e) PVA 6%, (f) PVA 3% + CS 3%, (g) PVA 3% + CS 1.5%, (h) PVA 3% + CS 3% + NM 0.5% + CZ 1%.

An average FD of this polymer combination shows 240 nm in another study, which is a higher value. , These FDs resulted in a voltage of 21 kV, FR of 0.2 mL/h, and a TCD of 9 cm. However, when the drugs are combined, bead formation can be observed. The presence of occasional bead-like structures in the PVA–CS–NM–CZ formulation can be attributed to several interconnected factors. The incorporation of dual drugs with different solubilities, hydrophilic neomycin (water solubility > 50 mg/mL) and hydrophobic clotrimazole (solubility ∼ 0.49 μg/mL in water), affects the solution rheology, surface tension, and electrical conductivity, which can lead to instabilities in the electrospinning jet and subsequent bead formation. Similar observations have been reported in other drug-loaded electrospun systems. Unnithan et al. observed bead formation when loading hydrophobic drugs into hydrophilic polymer matrices, attributing it to phase separation and localized solution property variations. Kenawy et al. reported that dual-drug loading can create heterogeneous solution properties that affect jet stability during electrospinning. The insolubility of clotrimazole in water, which is the main solvent of the polymer suspension, results in some undissolved drug particles remaining as solids in the polymer matrix, which ultimately develop into beads during the electrospinning process. Despite the presence of some beads, the overall fibrous morphology is maintained, and the material retains its functionality for wound dressing applications. Interestingly, these beads may actually serve as drug reservoirs, potentially contributing to sustained release kinetics and prolonged therapeutic effects. The SEM images confirm fiber formation with considerably sized pores, even with the beaded structure, which is advantageous for gas transmission and wound exudate management. With the same electrospinning parameters, an NM and CZ combined electrospun wound dressing has been fabricated. The bead formation that was observed upon introducing the drugs into the polymer matrix can be attributed to the fact that the insolubility of the drug CZ in water, which is the main solvent of the polymer suspension, leaves some undissolved drug as solid in the polymer matrix, which, along with the spinning process, ultimately will develop into beads. On the other hand, SEM images show fiber formation with considerably sized pores, even with the beaded structure, which is advantageous for gas transmission of the wound.

2.2.3. Mechanical Property Analysis

The tensile test of PVA, PVA–CS, and PVA–CS–NM–CZ films resulted in values of tensile strengths of 7.34, 7.02, and 7.49 MPa, respectively. Load at break was recorded as 7.05, 6.46, and 4.4 N for PVA, PVA–CS, and PVA–CS–NM–CZ films. Stress vs strain curves of (a) PVA, (b) PVA–CS, and (c) PVA–CS–NM–CZ films are shown in Figure . All the other details relevant to mechanical property analysis are summarized in Table below. The mechanical strength was evaluated through tensile testing, which yielded varying tensile strength values. Assessment of tensile strength is very important when it is focusing on the application of this electrospun film. As the target application of the fabricated electrospun membrane is to be used as a wound dressing, the mechanical strength of the nanofiber mat is critical, and it depends upon its intended application purpose and anatomical location. For general application as wound dressing, tensile strengths of 2–10 MPa are acceptable, but dressings applied to high-stress locations require higher mechanical properties. However, values (7.34 MPa for neat PVA, 7.02 MPa for the PVA–CS control, and 7.49 MPa for drug-loaded films) that are determined by this study are comparatively higher when compared with previous studies, which are also within the acceptable range for application as wound dressing. Comparative literature analysis proves enhanced mechanical performance: Zhang et al. achieved tensile strengths of 2.8–5.5 MPa for PVA/chitosan electrospun membranes, while Sudheesh Kumar et al. achieved 4.2–6.1 MPa for similar formulations. Furthermore, the neat PVA exhibits higher elongation, whereas the drug-loaded film displays high strength with low elongation. Hence, the mechanical strengths reported in this study can be considered comparable and better than those of the previous study.

3.

Stress vs strain curves of (a) PVA, (b) PVA–CS, and (c) PVA–CS–NM–CZ films.

2. Details of Tensile Test.

film	tensile strength (MPa)	load at break (N)	MOD 100% (MPa)
PVA	7.34	7.05	7.05
PVA–CS	7.02	6.46	6.91
PVA–CS–NM–CZ	7.49	4.49	-

Improved mechanical properties within this work are the result of optimal polymer ratios and process conditions. The negligible increase in tensile strength with drug loading (7.49 MPa) is evidence of optimal polymer–drug interactions that sustain the fiber matrix. Elastic moduli values (MOD 100%) of 7.05 MPa (PVA) and 6.91 MPa (PVA–CS) indicate good patient comfort and formability for contouring of the wound. They enable the dressing to withstand normal mechanical stresses with patient movement without being structurally compromised. Break load values (7.05 N for PVA, 6.46 N for PVA–CS, 4.49 N for drug-loaded films) are adequate for handling upon application. Reduction in drug-loaded films may be owing to sites of stress concentration created by drug particles, which is not a problem as tensile strength remains intact.

2.2.4. Porosity Measurement

The electrospun PVA, PVA–CS, and PVA–CS–NM–CZ films showed a porosity of 76.30 ± 3.15%, 71.35 ± 2.89%, and 65.30 ± 2.42% for (n = 3), respectively. The small standard deviation points to a fairly consistent fabrication process, with little variation between samples. This suggests that the electrospinning conditions were well controlled, leading to uniform fiber formation. The measured porosity is also comparable to values reported for similar electrospun wound dressings, supporting the presence of an interconnected pore network that is important for wound healing. The measured porosity confirms the successful formation of an interconnected porous network structure that is necessary for wound healing applications, and it falls within the reported range for similar electrospun wound dressing materials.

The results demonstrate that pure PVA electrospun mats exhibited the highest porosity at 76.39 ± 3.18% and upon incorporation of chitosan into the polymer matrix, the porosity decreased to 71.35 ± 2.95% for the PVA–CS blend, representing a 6.6% reduction compared to pure PVA. The subsequent loading of neomycin (0.5% w/v) and clotrimazole (1% w/v) further reduced the porosity to 65.30 ± 2.42% in the final PVA–CS–NM–CZ formulation, corresponding to an additional 8.5% decrease from the PVA–CS blend and an overall 14.5% reduction from pure PVA. The measured porosity of the electrospun PVA–CS–NM–CZ dressing was 65.30 ± 2.42%, which falls comfortably within the 60–90% range often cited as ideal for wound dressings and tissue engineering. , The relatively small standard deviations (2.42–3.18%) indicate good reproducibility of the electrospinning process and consistent porous structure formation across replicate samples. Porosity in this range is important because it supports several physiological processes needed for wound repair. Our results are consistent with earlier reports. For instance, Chong et al. (2007) reported that the electrospun scaffolds with porosity between 60 and 90% were well suited for tissue engineering. At 65.30%, the PVA–CS–NM–CZ composite provides a good balance between structural strength and biological function. This degree of porosity maintains interconnectivity of pores for oxygen, nutrients, and cell migration, while still ensuring enough fiber-to-fiber contacts for mechanical stability. This interconnected porous network of the electrospun wound dressing allows oxygen and nutrients to reach the wound bed, also allowing for the removal of waste, and encourages cell infiltration during tissue repair. In addition, the relatively high porosity enhances the dressing’s capacity to handle wound exudate, helping to prevent dehydration of dehydration while maintaining the moist environment required for effective healing. This feature is especially relevant for PVA–CS–NM–CZ composites, since both polymers are hydrophilic and naturally promote fluid absorption and retention. The decrease in porosity observed upon chitosan incorporation into the PVA matrix (from 76.39% to 71.35%, a 6.6% reduction) can be primarily explained by the strong intermolecular interactions between the two polymers. Both PVA and chitosan possess abundant hydroxyl (−OH) groups, and chitosan additionally contains amino (−NH₂) groups that can participate in extensive hydrogen bonding networks during the electrospinning process. , These hydrogen bonds create a more compact and dense fiber structure with reduced interfiber spacing, consequently decreasing the overall porosity of the nanofiber mat. The porosity value of 71.35% for the PVA–CS blend aligns well with previously reported values for similar polymer systems. A study by Venugopal et al. on chitosan-PVA fibrous wound dressings reported neat PVA mats with 74 ± 3.9% porosity and hybrid Ch–PVA mats with 52 ± 4.7% porosity. Another investigation on chitosan–PVA–silk composites showed Ch–PVA mats with 68 ± 4.4% porosity. Augustine et al. reported PVA–chitosan-neem extract blend nanofibers with 91% porosity, though this higher value likely reflects different polymer ratios and processing conditions. The results of this study fall within this reported range, confirming the reproducibility and validity of our fabrication method.

The further reduction in porosity to 65.30% upon incorporation of neomycin (0.5% w/v) and clotrimazole (1% w/v) into the PVA–CS matrix represents an additional 8.5% decrease and can be attributed to multiple interconnected mechanisms occurring during the electrospinning process and fiber formation. Both drug molecules are encapsulated within the polymer matrix during electrospinning, and their presence significantly influences the porous structure. Neomycin sulfate, being a hydrophilic aminoglycoside antibiotic with high water solubility, tends to distribute homogeneously within the hydrophilic PVA–chitosan blend. In contrast, clotrimazole, a hydrophobic imidazole antifungal agent with poor water solubility, may form microdomains or crystalline regions within the fiber structure. , These drug particles physically occupy spaces that would otherwise constitute pores, thereby reducing the overall porosity of the electrospun mat. The measured porosity has a direct impact on how cells interact with the wound dressing. Studies have shown that a porosity of 60–70% promotes optimal fibroblast attachment, growth, and migration. The porous nature mimics the native extracellular matrix (ECM) structure, which provides three-dimensional support for the cellular activities engaged in the healing of wounds. Furthermore, such a porosity level provides for the controlled release of bioactive compounds that could be incorporated into the PVA–chitosan matrix. Liquid displacement method using ethanol was an appropriate porosity measurement technique in electrospun PVA–chitosan mats. The miscibility of ethanol with PVA and chitosan polymers, as well as its low surface tension, guarantees complete wetting of the porous structure without dissolving or swelling the polymers. The high reproducibility (CV = 3.7%) confirms the stability of both the electrospinning process of fabricating the wound dressing material and the porosity measuring method. Such reproducibility is of critical importance to enable scale-up and clinical translation of the wound dressing material. The porosity of 65.30% of this PVA–chitosan electrospun wound dressing renders it very suitable for clinical use in wound healing. The porosity of the dressing plays an active role in all four stages of wound healing: hemostasis, inflammation, proliferation, and remodeling. In the early phases, the porous network supports blood clot formation and guides the migration of inflammatory cells. As healing progresses into the proliferative phase, an optimal level of porosity creates space for new blood vessels to grow and helps with the deposition of extracellular matrix components. The interconnected pores also ensure adequate oxygen delivery, which is essential for collagen synthesis and overall tissue regeneration.

2.2.5. Water Uptake Capacity

The water uptake capacity of PVA, PVA + CS, and PVA + CS + NM + CZ films was separately examined to identify and showcase the ability to absorb fluids, an essential wound dressing requirement. The swelling of each film in the considered time intervals and the weights of films at each time interval, concerning the weights of other time intervals, are represented in Table . The initial weight of films (a), (b), and (c) was 0.0248, 0.0248, and 0.0250 g, and those were weighed 0.0922, 0.0929, and 0.0998 g after swelling for 24 h, which resulted in a WUC of 271%, 275%, and 299% respectively. Enhanced water uptake of chitosan-based systems is attributed to the hygroscopic nature of chitosan and the availability of hydroxyl and amino groups in large numbers that hydrogen bond with water molecules. , The cationic character of chitosan under physiological pH creates electrostatic interactions with water, which facilitates the swelling capacity of the polymer matrix. WUC indicates that drug-loaded dressing absorbs more fluids compared to PVA and control films, which is approximately four times its initial weight after 24 h, which is advantageous in wound healing.

3. Water Uptake Capacity of PVA, Control, and Drug-Loaded Films.

	wet weight (g)
time (h)	(a) PVA 6%	(b) PVA 3% + CS 3%	(c) PVA 3% + CS 3% + NM 0.5% + CZ 1%
0.5	0.0777 ± 0.0003	0.0756 ± 0.0003	0.0747 ± 0.0003
1	0.0876 ± 0.0003	0.0770 ± 0.0002	0.0834 ± 0.0004
2	0.0881 ± 0.0002	0.0827 ± 0.0002	0.0913 ± 0.0003
4	0.0902 ± 0.0004	0.0880 ± 0.0004	0.0964 ± 0.0003
6	0.0919 ± 0.0003	0.0918 ± 0.0003	0.0990 ± 0.0005
24	0.0922 ± 0.0004	0.0929 ± 0.0003	0.0998 ± 0.0005

Drug-loaded films exhibited the highest water uptake (299.20 ± 3.03%) due to several reasons: (1) higher porosity created by drug particle-induced heterogeneity during electrospinning; (2) hydrophilic neomycin sulfate contributing extra water-binding sites; (3) clotrimazole particles, although hydrophobic in nature, acting as swelling promoters by creating microvoids that facilitate water uptake. The incorporation of drugs into the polymer suspension, followed by electrospinning, resulted in the formation of enlarged surface voids in the dressing, relative to the neat and control films. These morphological changes are likely to facilitate increased water absorption. This high absorption capacity is clinically beneficial in the management of exudates in highly draining wounds. The incremental increase from PVA (271.77 ± 2.10%) to PVA–CS (274.60 ± 3.27%) to drug-loaded films (299.20 ± 3.03%) demonstrates the additive characteristics of each component on fluid absorption capacity, supporting the formulation’s suitability to various types of wounds with varied levels of exudate. The water uptake capacity of each film is summarized in Table . Microscopic images of dry and swelled drug-loaded films are shown in Figure a–f and graphically represented in Figure h, showing the study of determining the water uptake capacity of each electrospun film before and after adding phosphate buffer saline (PBS).

4. Water Uptake Capacity.

film	initial weight (g)	final weight (24 h) (g)	WUC % (after 24 h)
PVA	0.0248 ± 0.0002	0.0922 ± 0.0004	271.77 ± 2.10
PVA–CS	0.0248 ± 0.0003	0.0929 ± 0.0003	274.60 ± 3.27
PVA–CS–NM–CZ	0.0250 ± 0.0003	0.0998 ± 0.0005	299.20 ± 3.03

4.

Water uptake capacity (a) microscopic image of dry (PVA–CS–NM–CZ) film, (b) microscopic image of (PVA–CS–NM–CZ) film after 30 min, (c) Microscopic image of (PVA–CS–NM–CZ) film after 1 h, (d) microscopic image of (PVA–CS–NM–CZ) film after 2 h, (e) microscopic image of (PVA–CS–NM–CZ) film after 4 h, (f) microscopic image of (PVA–CS–NM–CZ) film after 6 h, (g) microscopic image of (PVA–CS–NM–CZ) film after 24 h, (h) graphical representation of water uptake capacities of PVA film, PVA–chitosan film (PVA–CS), and PVA–chitosan–neomycin–clotrimazole film (PVA–CS–NM–CZ).

2.3. Pharmaceutical Studies

2.3.1. Drug Release Study

For the quantification of the NM drug released, UV–vis spectroscopy was used. The concentrations of drug released, measured at the λ_max of 307.5 nm at different time intervals, are summarized in Table . The concentration of the released drug during the first hour is 4.74 mg/mL, whereas the concentration after 24 h is 0.88 mg/mL. A higher drug concentration is released in the first hour, and at the end of 24 h, a cumulative amount of 11.88 mg was released to the system Figure . Absorbances of each time interval show a continuous reduction in the concentration, where 40% of the total NM was released during the first hour.

5. UV Data of NM Released after 1, 2, 4, 7, and 24 h.

time (hours)	absorbance	concentration (μg/mL)	total drug release (mg)	cumulative drug release (mg)
0	0.000	0.00	0.00	0.00
1	0.078	474.03	4.74	4.74
2	0.0535	325.14	3.25	7.99
4	0.0345	209.67	2.10	10.09
7	0.0150	91.16	0.91	11.00
24	0.0145	88.12	0.88	11.88

5.

Comparison of drug release of (i) neomycin, and (ii) clotrimazole using (a) cumulative drug release, (b) zero order kinetics, (c) first order kinetics, (d) higuchi model, (e) Kosmeyer-Peppas model, and (f) Weibull model.

Moreover, a high-performance liquid chromatography (HPLC) analysis was conducted to study the CZ release from the electrospun wound dressing. The peak areas of HPLC chromatograms are used to determine the concentration of samples at each time interval. The concentrations of each time interval are summarized in Table . After 24 h, a total of 14.33 mg was released into the system. The total drug release (mg) is calculated for 10 mL, which is considered the release system. HPLC peak areas of the CZ series show a clear reduction when the concentration is reduced. The HPLC chromatogram shows that 21.5% of the drug was released after 1 h, and after 2 h, it has increased to 51%. However, after 10 h, the peak area has reduced, and it shows a deceleration of drug release compared to the two earlier samples. Which is 79% of the drug released within the first 10 h. After 24 h, the peak area is further reduced. With this observation, it can be stated that CZ release increases gradually after a certain point, the release decreases as the CZ in the dressing releases continuously, and it can be expected a reduction in concentration with time. The total drug release in the considered period has been recorded as 11.88 mg and 14.32 mg of NM and CZ, respectively, Figure .

6. HPLC Data of CZ Release after 1, 2, 10, and 24 h.

time interval (hours)	retention time (min)	concentration (ppm)	total drug release (mg)	cumulative drug release (mg)
0	0	0	0.000	0.000
1	4.853	300.08	3.08	3.08
2	4.853	377.70	4.24	7.31
10	4.848	401.49	4.02	11.33
24	4.848	423.55	3.00	14.33

According to the drug release kinetics study, the clotrimazole-incorporated membrane followed the Higuchi model with a high regression coefficient (R ² = 0.97). In contrast, the neomycin-incorporated membrane followed the Weibull model with a lower regression value (R ² = 0.88). The Higuchi model describes Fickian diffusion-controlled drug release from a porous matrix. , Therefore, the excellent fit of clotrimazole’s release to the Higuchi model suggests that the formulation enabled the diffusion of this poorly water-soluble, hydrophobic drug (solubility ∼ 0.49 μg/mL in water) by likely embedding the drug in a hydrophilic or solubilizing matrix. This behavior suggests that clotrimazole may have been effectively encapsulated or dispersed within a capping or carrier agent used during membrane fabrication, thereby facilitating its pseudohydrophilic release profile.

In contrast, neomycin, a highly hydrophilic molecule (water solubility > 50 mg/mL), would typically be expected to follow a diffusion-based release mechanism (e.g., Higuchi). However, its release was better described by the Weibull model, , indicating a non-Fickian or complex release behavior, exhibiting matrix erosion or swelling. This deviation implies that the neomycin may be trapped or immobilized within the cross-linked polymer matrix, resulting in a sustained or slow release. , This could be due to strong ionic or hydrogen bonding interactions between neomycin’s multiple amine/hydroxyl groups and the matrix-forming agents. Considering the application of said membranes in clinical treatments at a physiological pH of wounded or open skin (typically around pH 7.5–8.5), the differential release profiles observed suggest that the membrane formulations were successfully optimized. Specifically, they enhanced clotrimazole release despite its hydrophobic nature, while effectively controlling the burst release of neomycin, providing a controlled release for both drugs.

This research establishes that clotrimazole and neomycin are both characterized by high burst release profiles, with the majority of the drug released within the initial ∼10 h. The quantified dual-phase drug release profiles exhibit significant therapeutic advantages for wound healing therapies. However, the benefits and drawbacks of this rapid release profile in wound healing applications must be carefully considered. Rapid antimicrobial intervention within the first 24 h has been shown in studies to significantly improve healing outcomes by reducing infection-related complications and preventing the formation of bacterial biofilm. That initial rapid release of neomycin (40% in the first hour) provides instant antimicrobial action crucial for inhibiting biofilm formation during the critical early stages of wound healing. The burst release profile is consistent with clinical needs for rapid reduction of bacterial loads, which is very significant in contaminated wounds where delayed antimicrobial activity can lead to systemic complications. The rapid release of the drug corresponds with the inflammatory phase of wound healing, which typically lasts from day 0 to day 3. During this period, a quick antimicrobial effect is crucial to limit infection and support the transition into the proliferative phase. Maintaining an aseptic environment in the wound at this early stage helps trigger the healing cascade effectively, aided by the higher initial concentration of the drug. As reported by Velnar et al. (2009), appropriate management of the inflammatory phase is critical, since the load of bacteria strongly influences the progression of later stages of healing. Conversely, the rapid release of neomycin has potential deficiencies as well, which are discussed here. The initial high concentration level (4.74 mg/mL) may cause local irritation of sensitive tissues in patients, and the reduction to 0.88 mg/mL after 24 h raises questions about maintaining the therapeutic concentrations over an extended period. Rapid drug release can lead to early depletion from the dressing, which may leave the wound susceptible to reinfection. Our findings indicate that within 24 h, the drug concentration falls to levels that are likely inadequate for continued antimicrobial protection. This limitation is especially concerning in chronic wounds, where impaired host defenses demand longer-lasting antimicrobial coverage. Due to this rapid drug depletion, it may require more frequent dressing changes, which not only raises treatment costs but also adds to patient discomfort. As Abrigo et al. (2014) point out, a major goal of advanced wound dressings is to reduce the need for frequent changes, an advantage that is undermined when the drug is exhausted too quickly.

The sustained release profile of Clotrimazole (21.5% at 1 h to 79% at 10 h) provides sustained antifungal protection essential for the avoidance of opportunistic fungal infections that typically develop 48–72 h after initial wound contamination. The release follows the typical onset time of fungal infection in chronic wounds. The delivery mechanism is by Higuchi-type diffusion, ensuring consistent drug availability during the important periods of wound healing. The complementary release patterns yield a therapeutic window with high bacterial killing in the early stage and enduring antifungal protection. This approach reverses the temporal sequence of microbial colonization of wounds in chronic wounds. Clinical comparative trials with commercial antiseptic dressings show dual-drug systems have 85% more efficient rates of wound closure compared to single-agent treatments. Potential limitations are the potential for drug interactions and dressing changes before total surgical drug depletion. Future development of formulations should focus on prolonging neomycin release duration with no adverse effect on the useful clotrimazole profile.

2.3.2. MTT Assay

The results of the MTT assay show exceptional biocompatibility of both drug-loaded and nondrug-loaded PVA–CS electrospun wound dressings across all tested concentrations (0.195–25 mg/mL) in both Vero and BHK cell lines. Vero cells have maintained 100 ± 2–5% viability for nondrug-loaded (control) samples and 99.52–100 ± 4–15% for drug-loaded samples across all concentrations. BHK cells have demonstrated 100 ± 0–3% viability for the nondrug-loaded (control) sample and a viability of 100 ± 0–2% for drug-loaded samples. Slightly higher standard deviations were observed in drug-loaded Vero samples at lower concentrations (11–15% at ≤1.5625 mg/mL) compared to higher concentrations (2–4% at ≥3.125 mg/mL), which may reflect variability in drug release. This is represented graphically in Figure . Drug-loaded and nondrug-loaded (control) formulations do not differ statistically significantly in either cell line at any concentration. According to ISO 10993-5 criteria, all formulations were deemed noncytotoxic (viability > 70%), surpassing the threshold by roughly 30 percentage points, suggesting a broad safety margin for clinical use.

6.

Graphical representation of results of cell viability by MTT assay for control and drug-loaded electrospun films (a) against vero cells, and (b) against BHK cells.

The excellent biocompatibility of this study (99.5% cell viability) greatly exceeds ISO 10993-5 requirements (>70%) and is in line with previous studies on PVA/chitosan-based systems. Adeli et al. (2019) reported that L929 fibroblasts exposed to PVA/chitosan/starch electrospun mats demonstrated 72%−90% viability. Bukar et al. (2024) found that green-synthesized alumina nanoparticles in vero and LT cells had IC₅₀ values of 153.3 and 252.0 μg/mL after 24–48 h, with 75% viability maintained even at 480 μg/mL. The superior safety profile of our controlled release electrospun system, compared to direct drug exposure, is supported by findings from Correa et al. (2018). In their study, fluconazole exhibited marked cytotoxicity in vero cells, with an IC₅₀ of approximately 1306 μM and only 35.25% cell viability at 2612.1 μM after 24 h. In contrast, the results from the present study demonstrate a markedly improved safety profile using the controlled release electrospun formulation. Nitanan et al. (2013) similarly found that neomycin-loaded PVA nanofibers exhibited reduced cytotoxicity compared to free neomycin through controlled release mechanisms. The concentration-independent biocompatibility (0.195–25 mg/mL) provides margins of safety, in contrast with concentration-dependent effects in chemically synthesized antimicrobial systems. Vero (epithelial) and BHK (fibroblast) cell lines have been selected to provide complementary safety information relevant to wound healing, as both cell types are crucial in re-epithelialization and tissue regeneration. PVA and chitosan’s inherent biocompatibility, FDA approval status, and chitosan’s properties of wound healing (hemostatic, anti-inflammatory, antimicrobial) are aspects that contribute to the observed safety profile. The slightly higher variability in drug-loaded vero samples at lower concentrations (11–15%) could be reflective of drug distribution heterogeneity within electrospun fibers or fiber–cell interaction dynamics, as referenced by Bukar et al. (2024), though mean viability was ≥99%.

2.3.3. Antibacterial Study

This study examined the antibacterial properties against one Gram-positive and one Gram-negative bacteria using the disk diffusion method mentioned in the analysis section. The zone of Inhibition (ZOI) was determined by measuring the diameter in millimeters. A small diameter indicates low antibacterial activity, whereas a larger one indicates higher antibacterial activity. ZOI values are summarized in Table . The control film sample (PVA 3% + CS 3%) shows 6 mm ZOI against S. aureus and E. coli. The drug-loaded film sample (PVA 3% + CS 3% + NM 0.5% + CZ 1%) shows 22.5 ± 0.7 mm and 18.5 ± 0.7 mm ZOI against S. aureus and E. coli, respectively. ZOI of both the control film sample (marked as (−) and the drug-loaded film sample marked as (+)) (in the same petridis) can be seen in Figure a–c. It is observed that a higher antibacterial property of drug-loaded electrospun wound dressing and a slight antibacterial property toward both Gram-positive and Gram-negative bacteria. Different studies indicate different values of ZOI for both bacterial groups. ,, With the ZOI values received for this study, it can be stated that fabricated drug-loaded films show significant antibacterial properties. Similar antibacterial studies have been conducted to examine the antibacterial properties of developed wound dressings previously and have reported different ZOIs. In a study where an electrospun wound dressing was developed, the ZOI against S. aureus was recorded as 7.8 mm to 8.9 mm for various formulations. With the comparison of them, the ZOI value that was recorded in this study is high in magnitude.

7. Zones of Inhibition of Bacterial Study.

		zone of inhibition (mm)
bacteria	bacteria type	control film (−)	drug loaded film (+)
Staphylococcus aureus	gram positive	6 ± 0	22.5 ± 0.71
Escherichia coli	gram negative	5.8 ± 0.35	18.5 ± 0.71

2.3.4. Antifungal Study

This study was conducted to determine the antifungal properties of electrospun films. After 48 h, Lasiodiplodia theobromae was grown in the whole plate while showing a clear ZOI around the drug-loaded sample disk with a mean diameter of 27.8 ± 1.7 mm and not showing any ZOI around the control film sample. Mean was calculated by getting four measurements from two plates. ZOI of the drug-loaded film sample against Aspergillus niger was 35.8 ± 7.4 mm, and the nondrug-loaded film (control film sample) showed no antifungal activity after 5 days. ZOIs of control film samples against both fungi groups are 0 mm. ZOIs are summarized in Table and represented in Figure a–c. When comparing the values with similar studies, it can be stated that fabricated drug-loaded electrospun wound dressing showcases antifungal properties , which suggests promising wound healing with this wound dressing.

8. Zones of Inhibition of Fungal Study.

	zone of inhibition (mm)
fungi	control film	drug loaded film
Aspergillus niger	0	27.8 ± 1.71
Lasiodiplodia theobromae	0	35.8 ± 70

7.

Zones of inhibition of antimicrobial study (a) ZOI of Staphylococcus aureus, (b) Escherichia coli, (c) ZOI comparison of the antibacterial test (d) ZOI of Aspergillus niger, (e) Lasiodiplodia theobromae, and (f) ZOI comparison (drug-loaded film sample) of antifungal test.

3. Conclusion

This study successfully designed and described a new multifunctional electrospun wound dressing that addresses the urgent need for polymicrobial infection of chronic wounds. The key innovation is the concerted manipulation of immediate antibacterial activity via neomycin release and sustained antifungal defense via controlled clotrimazole diffusion in a biocompatible PVA/chitosan matrix. This research work demonstrates several successful outcomes, which are, successful optimization of electrospinning parameters to fabricate uniform nanofibers with controlled drug loading, the generation of complementary drug release kinetics with targeted wound healing phases, and the achievement of enhanced antimicrobial activity versus both bacterial and fungal pathogens dominant in chronic wounds. The MTT assay confirmed excellent biocompatibility with cell viability exceeding 99% across all tested concentrations, demonstrating the materials are noncytotoxic and safe for potential application. However, the MTT assay evaluates cytotoxicity rather than direct wound healing capability. Clinical significance of this work lies beyond traditional single-agent therapy with its provision of a broad antimicrobial approach that may reduce healing times, reduce dressing changes, and improve patient outcomes for chronic wound management. The high cytocompatibility and mechanical properties also justify its clinical translatability. Future research needs to be directed toward in vivo validation in controlled animal models to quantify the rates of wound healing, safety profiles, and best dosing regimens. Clinical trials will ultimately be needed to confirm how effective the dressing is in real patients and to define its role within current wound care practices. Alongside this, developing reliable large-scale manufacturing methods and assessing cost-effectiveness will be key steps toward bringing the product into healthcare systems. Overall, this multipurpose dressing represents an exciting advance in wound care, with significant potential to address the challenges of managing chronic wounds in modern medicine.

4. Materials and Methods

4.1. Materials

For the polymer suspension preparation, PVA was obtained from Himedia (M _W = 60,000 – 125,000), chitosan (80% deacetylated and high molecular weight), methanol, and glacial acetic acid were obtained from Sigma-Aldrich. Samples of neomycin sulfate (NM) and clotrimazole (CZ) were received by ASTRON LIMITED, Sri Lanka. Tween 80 was purchased from a local chemical shop. A phosphate buffer solution (PBS) is made by using KH₂PO₄ and K₂HPO₄.

4.2. Preparation of Polymeric Solution

PVA 3% (w/v) solution was made by using 0.30 g in 9.70 mL of distilled water at 80 °C. The clumps of PVA were dissolved by mixing with spatulas and stirred continuously at 400 rpm at the same temperature. After obtaining a clear solution, the temperature was reduced to 40 °C and stirred at 250 rpm overnight. A CS 3% (w/v) solution made in 70% (v/v) acetic acid. In this step, the initially used 6% (w/v) total solid loading was achieved by combining 3% (w/v) of chitosan and 3% (w/v) of PVA. Afterward, chitosan concentration was reduced gradually while keeping the PVA 3% (w/v) concentration constant. An amount of 0.30 g of CS was measured and added dropwise with continuous stirring at 400 rpm in 9.70 mL of 70% (v/v) acetic acid solution at a temperature of 40 °C. The chitosan solution was stirred at 250 rpm and at room temperature overnight. PVA and CS solutions were added together in 80% (v/v) and 20% (v/v) ratios and stirred for 4 h at room temperature. Next, NM, 0.05 g, was weighed and dissolved in 1 mL of distilled water, and was slowly added to the PVA + CS solution. Further, CZ 0.10 g was weighed and dissolved in 500 μL methanol, Tween 80 (50 μL, approximately 1–2 droplets), and 500 μL of distilled water. After mixing, the CZ suspension was added to the PVA + CS + NM solution and allowed to stir for another 4 h at room temperature. After that, 10 mL of PVA + CS + NM + CZ solution was sonicated in a bath sonicator for 15 min at room temperature to remove air bubbles.

4.3. Electrospinning of the Polymeric Solution

The electrospinning unit is set up with 3 main parts: a high-voltage power supply, the syringe pump, and the stable collector. The high-voltage power supply (MATSUSADA Precision Inc.) was used to supply a high voltage. The positive and negative terminals were connected to the syringe tip and the metallic collector. The ground terminal is connected to a grounding plug base. A syringe pump (Thermo Scientific Chemyx Fusion 100t Dual Pump) was used to pump polymer suspension. A polymer suspension filled a 10 mL disposable syringe with a flat tip needle (needle gauge 18G) was clamped in the syringe pump. The stable steel metallic collector was covered with aluminum foils. The syringe pump is powered on, and the following specifications were inserted: syringe and its internal diameter (10 mL plastic), filled polymer volume (10 mL), and rate (0.2 mL/h). The pump with a syringe was placed inside the chamber while maintaining a tip-to-collector distance (TCD) of 9 cm. A stable collector was kept parallel to the wall of the chamber. Then the pump was started, and the high-voltage power supply was switched on. The voltage was optimized before and set to 21 kV. Electrospun fibers were collected from the aluminum foil, dried in a vacuum oven for 6 h at 40 °C, carefully removed from the foil using forceps and stored in airtight containers. The same procedure was repeated at each electrospinning trial while varying the polymer solution composition and the electrospinning parameters.

4.4. Characterization of Electrospun Nanofibers

The fabricated film is subjected to characterizations to examine its physical, chemical, and pharmaceutical properties.

4.4.1. Fourier Transformation Infrared Spectroscopy

FT-IR is a vibrational spectroscopy technique that is used in chemical characterization. It provides information about types of bonds, functional groups, and integrated molecule confirmation. FT-IR (PerkinElmer Spectrum 2) was used, and the spectrum was recorded between 500 cm^–1 and 4000 cm^–1 in the ATR mode. The drug-loaded electrospun film, NM, CZ, CS, and PVA in their original forms, are examined through FTIR. The samples were in powder form (API) and electrospun thin films.

4.4.2. Scanning Electron Microscopy

Scanning electron microscopes (Hitachi SU6600 FE-SEM and ZEISS model SEM) were used to examine the morphology of the electrospun film. SEM uses a focused electron beam on the specimen. Samples were stuck to stubs using carbon tape and coated with gold sputtering to observe the sample’s morphology using SEM at 10 kV and 8 kV accelerating voltages. These average fiber dimensions were analyzed using SEM images. The average fiber diameter dimensions of approximately 100 points were taken, and the average fiber diameters were determined by ImageJ software and displayed (histograms) using the Origin Pro student version.

4.4.3. Mechanical Properties

A tensile test is conducted to examine the mechanical properties of electrospun PVA 6% film, 3% PVA + 3% CS film, and 3% PVA + 3% CS + 0.5% NM + 1% CZ film, such as tensile strength, elastic modulus, and load at the break by using INSTRON 3365 UTM. The specimens were cut into bumble shapes with 50 ± 10 mm, and three samples were tested from each sample type. This test was conducted according to the ASTM D412 standard.

4.4.4. Porosity Measurement

The porosity of the electrospun PVA, PVA–CS, and PVA–CS–NM–CZ nanofibrous mats was determined by the liquid displacement method with ethanol as the displacement fluid, as per the procedure described by Arampatzis et al. (2021). The method was adopted because it is nondestructive, simple, and hydrophilic polymer matrices-friendly, such as PVA–chitosan composites. Circular disc samples of PVA, PVA–CS, and PVA–CS–NM–CZ (drug-loaded) electrospun films were cut out from the electrospun mats for porosity determination. For this, three (n = 3) electrospun film samples from each type were randomly selected, and each sample was precisely weighed by an analytical balance (±0.1 mg accuracy) to approximately 25 mg. Each sample was then carefully added to a graduated cylinder containing 7 mL of ethanol (V ₁). Volume after the samples were immersed was recorded as V ₂. Samples were left for 10 min to permit complete penetration of the porous network by ethanol. After the equilibration time, the fiber mat was carefully removed with tweezers, and the volume of the residual ethanol was measured as V₃. Then the percentage of porosity was calculated using the following formula (eq ).

$$\mathrm{P}\mathrm{o}\mathrm{r}\mathrm{o}\mathrm{s}\mathrm{i}\mathrm{t}\mathrm{y}(\%)=[(V_1-V_3)/(V_2-V_3)]\times 100$$ 1

V ₁ = initial ethanol volume. V ₂ = volume after sample immersion. V ₃ = final volume after sample removal.

Each sample was tested in triplicate (n = 3) to improve statistical reliability, and the results are presented as mean ± standard deviation. All measurements were carried out at room temperature to ensure consistent experimental conditions.

4.4.5. Study of Water Uptake Capacity

A swelling test was conducted to determine the ability of the electrospun films to uptake fluids. Typically, a wound generates fluids, and a wound dressing should be able to absorb those. Here, patches of the electrospun films in triplicate (n = 3) for each film type were soaked in PBS to examine swelling properties. Patches weighing approximately 25 mg were submerged in Petri dishes and allowed to swell. At time intervals of 0.5, 1, 2, 4, 6, and 24 h, samples were taken out, and surface water was removed using a filter paper, and the weight was measured. Standard deviations were calculated at each time point to impart statistical reliability. At the same time intervals, light microscopic images were captured by a Nikon ECLIPSE Ni light microscope to examine the swelling behavior of drug-loaded PVA + CS + NM + CZ (3% + 3% + 0.5% + 1%) film. The water uptake capacity (WUC) was calculated by using eq . The water uptake capacity study was conducted on neat PVA (6%) film, PVA + CS (3% + 3%) film, and drug-loaded PVA + CS + NM + CZ (3% + 3% + 0.5% + 1%) film.

$$\mathrm{W}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{r}\mathrm{u}\mathrm{p}\mathrm{t}\mathrm{a}\mathrm{k}\mathrm{e}\mathrm{c}\mathrm{a}\mathrm{p}\mathrm{a}\mathrm{c}\mathrm{i}\mathrm{t}\mathrm{y}(\%)=[(W_2-W_1)/(W_1)]$$ 2

W ₁ = initial weight. W ₂ = final weight.

4.5. Pharmaceutical Studies

4.5.1. Drug Release Study

A PBS was made as the release medium for NM using KH₂PO₄ and K₂HPO₄ with a strength of 100 mmol. A series of NM solutions was made in 100, 200, 300, 400, and 500 μg/L concentrations. The calibration curve for the NM series was constructed by measuring the absorbance of each concentration at 307.5 nm and by plotting the absorbance versus concentration graph. Afterward, the release study was conducted. A patch of drug-loaded electrospun film weighing 25 mg was immersed in a beaker filled with 10 mL of PBS at 37 °C and kept on the shaker at a speed of 120 rpm, and the temperature was kept constant at 37 °C. A temperature of 37 °C is considered the normal body temperature of a human. At the time intervals of 1, 2, 4, 7, and 24, 2 mL each was removed from the beaker and stored separately, and at each instance, 2 mL of fresh PBS was returned to the system to keep the volume constant. After collecting all the samples, the absorbances were recorded. Values were taken from two identical systems.

HPLC was conducted to study the release of CZ from the film. This study followed a similar study. The mobile phase was used as a combination of methanol and K₂HPO₄ (0.0125 M) in a ratio of 85:15. A C18 column was used as the stationary phase, and a flow rate of 1.5 mL/min. The detection wavelength was set to 254 nm, and the glass syringe was injected with a sample of 20 μL volume. A 25, 50, 75, and 100 ppm of CZ series was analyzed to construct the calibration curve by peak area versus concentration. The samples were stored in glass vials and filtered by a disposable 0.45 μm pore-sized syringe filter (one for each sample) separately. HPLC was purged and the mobile phase ratios were entered into the machine as 85:15 of methanol and K₂HPO₄. A sample at a time was injected at the injection position and turned to the lock position. The total retention time was set at 10 min. Samples were collected in 1, 2, 10, and 24 h to measure the release by HPLC. To interpret the values and construct graphs, MS Excel and Origin Pro (student version) were used.

4.5.2. MTT Assay

An MTT assay was conducted to evaluate the cytotoxicity of both drug-loaded and nondrug-loaded electrospun films. For that, Vero cells, which are kidney epithelial cells derived from the African green monkey, and BHK-21 cells, which are fibroblast-like cells derived from baby hamster kidney tissue, have been selected to provide complementary safety information relevant to wound healing. Both cell lines were maintained in Dulbecco’s modified Eagle medium (DMEM; Sigma-Aldrich, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Sigma-Aldrich, USA) and 1% (v/v) penicillin–streptomycin antibiotic solution. The cells were cultivated at 37 °C in a humidified environment with 5% CO₂ in a standard cell culture incubator. Cells were detached with 0.25% trypsin–EDTA and seeded into 96-well plates at 5000 cells/well in 180 μL complete medium. After 24 h of incubation, cells were treated with 20 μL of test materials. 25 mg of the test material was dissolved in 1 mL of saline, vortexed thoroughly, and placed on a shaker for 24 h. The resulting extract was filtered and subsequently used for the cell culture experiments (the extracts of drug-loaded film (3% PVA + 3% CS + 0.5% NM + 1% CZ), and nondrug-loaded film (3% PVA +3% CS film)) to obtain a final volume of 200 μL per well. Untreated cells were used as the control. Treatments were tested at a concentration range of 25.0–0.195 mg/mL, serial dilutions of 25.0, 12.5, 6.25, 3.125, 1.5625, 0.7813, 0.3906, and 0.195 mg/mL. After 24 h, 10 μL of MTT stock solution (5 mg/mL in PBS) was added to each well, and the plates were incubated for 2 h at 37 °C. Then, after the incubation, the media were carefully aspirated, and 100 μL of DMSO was added to each well to dissolve formazan crystals. Absorbance was measured at 570 nm using a microplate reader (BioTek). The percent viability was calculated by the following eq . Data are presented as mean ± SD from at least three independent experiments.

$$\mathrm{C}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{v}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}(\%)=[(A_{570}\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e})/(A_{570}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l})\times 100]$$ 3

A ₅₇₀ sample = absorbance of extract-treated cells. A ₅₇₀ control = absorbance of untreated control cells.

4.5.3. Antibacterials Study

The antibacterial test was conducted to determine the antibacterial properties of the fabricated electrospun films. The drug-loaded film (3% PVA + 3% CS + 0.5% NM + 1% CZ) and control film (3% PVA + 3% CS film) were subjected to this study by disk diffusion method on one Gram-positive bacteria, S. aureus (ATCC 25923), and one Gram-negative bacteria, E. coli (ATCC 25922), according to the Hudzicki J. Kirby–Bauer Disk Diffusion Susceptibility Test Protocol of 2016, American Society for Microbiology. Mueller–Hinton agar plates were used to grow bacteria, and by using sterilized swabs, an inoculation procedure was done. Disks of both drug-loaded film and nondrug-loaded film (control) with a 6 mm diameter were placed on inoculated agar plates and observed for 24 and 48 h. Two triplicates from each test were conducted. Then, the zones of inhibition (ZOI) were measured using a ruler or a caliper.

4.5.4. Antifungal Study

The antifungal test was conducted to determine the antifungal properties of the fabricated electrospun films. The drug-loaded film (3% PVA + 3% CS + 0.5% NM + 1% CZ) and the control film (3% PVA + 3% CS film) were subjected to this study. This test was conducted in the disk diffusion method on two fungal groups, A. niger (ATCC16888), and L. theobromae (MT990527). The premade and autoclaved potato dextrose agar (PDA) plates were used to grow the fungi. The disks with a diameter of 6 mm of both samples and a disk shallow with distilled water were placed on the plate, and the fungi sample was placed in the center of the plate. Plates were closed with new Petri dishes and sealed in polythene bags. L. theobromae is fast fast-growing fungus; therefore, the ZOI was measured within 48 h, and the ZOI of A. niger was measured after 5 days. The mean ZOI was obtained in millimeters. Two triplicates from each test were conducted.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c07024.

• The Supporting Information contains details related to the optimization of electrospinning parameters, polymer suspension preparation, and electrospinning conditions (Tables S1 and S2). It also includes additional SEM images of electrospun fibers at various magnifications, as well as at each parameter change, featuring PVA/chitosan blend fibers and drug-loaded formulations (Figures S1–S4) (PDF)

The authors declare no competing financial interest.